Methods and compositions related to long half-life coagulation complexes

ABSTRACT

Disclosed herein are methods and compositions related to coagulation factor complexes comprising a coagulation factor; a fusion protein; and a modifying molecule, wherein the modifying molecule is coupled to the coagulation factor in such a way as to allow binding by the fusion protein, thereby creating a modified coagulation factor; wherein the modilied coagulation factor and the fusion protein interact in at least two independent sites.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication No. 62/203,973, filed Aug. 12, 2015, which is incorporatedherein by reference in its entirety.

BACKGROUND

Blood coagulation is controlled by a very complicated series of checksand balances such that coagulation only is triggered in the event of ableed (Smith, Travers, & Morrissey, 2015). Injury sets off activation ofthese enzymes, resulting in an amplifying cascade of reactions thatseals the wound. Hemophilia results from a defect in a gene coding forone of these proteins such that the cascade is aborted prematurely andbleeding continues. The most common forms of hemophilia, hemophilia A,hemophilia B and von Willebrand's disease, have long been treated byinfusion of purified factor concentrates, replacing the defective enzymeand restoring the ability of the blood to clot.

Infusion of factor is remarkably effective, allowing afflictedindividuals who may have died in childhood to have normal lifeexpectancies (Hoots, 2003). With the increasing use of prophylaxis, thatis, regularly scheduled infusions of factor to maintain a reasonablelevel of protection, these patients can lead essentially normal lives(Srivastava et al., 2012). This does not come without cost, literallyand figuratively. Patients with severe hemophilia A need to infusefactor every other day due to the short circulatory half life of FactorVIII (FVIII), the protein missing in that form of the disease. Thiscreates a number of problems, such as continued venous access andnoncompliance.

Another very serious problem is encountered when patients developinhibitory antibodies to the infused FVIII (Kempton & Meeks, 2014).About 30% of all hemophilia A patients will develop antibodies at somepoint in their therapy but about 5% develop such a serious inhibitorproblem that FVIII infusion is no longer effective. This necessitatesthe use of “bypass” therapy. Factor VIIa (FVIIa) is one of theinitiators of the coagulation cascade and can be used to step around theneed for either FVIII or Factor IX (FIX) in the process. This requiresvery high concentrations of FVIIa and very frequent dosing since FVIIahas a circulatory half life of only two hours.

Because of these and other reasons, longer half-life factors are verydesirable (Pipe, 2010). Less frequent dosing should improve compliance,venous access problems and expose the patient to a smaller mass ofpurified protein, perhaps reducing inhibitor formation. Moreover, longerhalf life proteins could expand treatment to the estimated 70% ofhemophilia patients worldwide who are still untreated. Cost of factor ismajor issue but so is the complicated medical service required forhemophilia patients, particularly children. Since factor needs to beinfused intravenously, rather than simply being injected subcutaneously,children with severe disease are most frequently treated at specializedhemophilia treatment centers. An obvious impediment to their treatmentis that they must be delivered to the center several times per weekwhich, in less developed countries, can put therapy beyond reach.Factors that persisted for longer periods of time could reduce thesetrips to once per week or even twice per month.

This problem has been recognized for some time and there have beennumerous attempts to prolong the half life of factors. There are twocommon strategies for increasing the half life of therapeutic proteins.The first is to modify the proteins with chains of polyethylene glycol,commonly called PEGylation (Ginn, Khalili, Lever, & Brocchini, 2014).The PEG chains increase the water of hydration around the protein whichresults in reduced affinity for certain receptors and antibodies. Thesecond strategy is to make use of the neonatal Fc receptor (FcRN) viafusion of the target protein with either the Fc portion of theimmunoglobulins or human serum albumin (Andersen et al., 2011). Bothimmunoglobulins and albumin have long circulatory half lives due totheir interaction with and protection by FcRN. When albumin orimmunoglobulins are internalized in a variety of cells, they bind toFcRN and are recycled to the surface rather than being degraded. Both ofthese proteins have half lives of several weeks as a result.

These strategies have been successfully utilized to increase the halflife of human Factor IX, the protein involved in hemophilia B (Mannucci& Mancuso, 2014). They have been less successful in prolonging the halflife of FVIII (Buyue et al., 2014; Stennicke et al., 2013). FVIII itselfis an unstable protein and requires the presence of von WillebrandFactor (vWF). FVIII in the absence of vWF has a half life of only a fewminutes. The half life of the complex is determined by the half life ofvWF so modifications to FVIII have only a small effect, increasinghalf-life from 12 hours to 18 hours.

Similar strategies have been attempted for FVIIa including PEGylation,fusion to albumin and Fc (Oldenburg & Albert, 2014; Schulte, 2008; vander Flier et al., 2015). Each of these modifications increased thehalf-life from 2 hours to over 10 hours but failed to solve anotherissue with FVIIa. FVIIa is most active in complex with Tissue Factor(TF). In the absence of TF, very high concentrations of FVIIa are neededto effect hemostasis. In some cases of engineered FVIIa, this hasresulted in inhibitor formation.

Accordingly, there is a need for compositions and methods for longhalf-life coagulation complexes.

SUMMARY

Disclosed herein is a coagulation factor complex comprising: acoagulation factor; a fusion protein comprising a first protein fused toalbumin, or an albumin fragment; and a modifying molecule, wherein themodifying molecule is coupled to the coagulation factor in such a way asto allow binding by the fusion protein, thereby creating a modifiedcoagulation factor; wherein the modified coagulation factor and thefusion protein interact in at least two independent sites.

Disclosed are kits comprising the coagulation factor complexes disclosedherein.

Also disclosed are methods of treating a subject with a diseaserequiring coagulation factor infusion, the method comprisingadministering to the subject the coagulation factor complex disclosedherein. The disease can be hemophilia, for example. The administrationof the coagulation factor complex to the subject result can result in ablood level half-life of the coagulation factor complex which is greaterthan the blood level half-life obtained upon administration of thecoagulation factor alone. The coagulation factor complex can beadministered to the subject via injection, inhalation, internasally, ororally.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 shows a scheme for the construction of a long half-lifecoagulation factor VIII (coagulation factor complex, including amodified coagulation factor).

FIG. 2 shows the schematic for construction of a long half-life, highactivity coagulation factor VIIa (coagulation factor complex, includinga modified coagulation factor).

FIGS. 3A and B show that the attachment of a modifying molecule to FVIIIdoes not interfere with its activity. FIG. 3A shows FVIII modified witheither maleimide-PEG1000-laurate or maleimide-PEG1000-fluoresceinisothiocyanate exhibit the same activity as the unmodified FVIII. FIG.3B shows untreated FVIII, FVIII subjected to the same experimentalconditions but without the modifying molecule and FVIII treated with themodifying molecule, maleimide-PEG1000-laurate, have the same activity.

FIGS. 4A and B show the modifying molecule attaches to the heavy chainof FVIII. FIG. 4A shows a silver stained SDS polyacrylamide gel (4 to20%) with untreated FVIII (Control) and modified FVIII (Modified FVIII).The modifying molecule is maleimide-PEG1000-biotin. FIG. 4B shows anelectroblot of a similar gel. The blot was probed with an avidin-horseradish peroxidase (HRP) then stained for HRP to visualize the modifyingmolecule.

FIGS. 5A-C show albumin binds to the modified FVIII. FIG. 5A shows FVIIIchromatographed on Superdex S200 Increase in 20 mM HEPES, pH 7.4, 150 mMNaCl, 4 mM CaCl₂, 0.01% Tween 20 and assayed for FVIII activity. FIG. 5Bshows modified FVIII (maleimide-PEG1000-laurate) preincubated with 10μg/ml human serum albumin before chromatography in the same buffer.Albumin shifts the molecular weight higher. FIG. 5C shows albumin doesnot affect the activity of modified FVIII.

FIGS. 6A-D show the structure of the fusion proteins and formation ofthe modified factor complex. FIG. 6A shows the structure and SDSacrylamide gel electrophoresis of the fusion protein monomer, CM110.FIG. 6B shows the structure and SDS acrylamide gel electrophoresis ofthe fusion protein dimer, CM210. FIG. 6C shows modified FVIII (modifiedwith either maleimide-PEG1000-laurate or maleimide-PEG1000-myristate)preincubated with 10 μg/ml of the fusion protein monomer (CM110) beforechromatography on Superdex S200 Increase in 20mM HEPES, pH 7.4, 150 mMNaCl, 4 mM CaCl₂, 0.01% Tween 20. FIG. 6D shows that the addition ofCM110 has no effect on the activity of modified FVIII.

FIGS. 7A-D show click chemistry to ligate modified FVIII to the fusionprotein. FIG. 7A shows the structure of the click chemistry reagentsused to modify FVIII. FIG. 7B shows maleimide-PEG4-6 methyl tetrazine(MPT) does not affect the activity of FVIII. FIG. 7C shows incubation ofMPT modified FVIII with die fusion protein CM110 modified withmaleimide-PEG3-trans cyclooctene, results in a high molecular weightcomplex that retains FVIII activity after chromatography on SuperdexS200 Increase in 20 mM HEPES, pH 7.4, 150 mM NaCl, 4 mM CaCl₂, 0.01%Tween 20. FIG. 7D shows formation of the complex does not affect FVIIIactivity.

DETAILED DESCRIPTION

The materials, compositions, and methods described herein can beunderstood more readily by reference to the following detaileddescriptions of specific aspects of the disclosed subject matter and theExamples and Figure included herein.

Before the present materials, compositions, and methods are disclosedand described, it is to be understood that the aspects described beloware not limited to specific synthetic methods or specific reagents, assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularaspects only and is not intended to be limiting.

Also, throughout this specification, various publications arereferenced. The disclosures of these publications in their entiretiesare hereby incorporated by reference into this application in order tomore fully describe the state of the art to which the disclosed matterpertains. The references disclosed are also individually andspecifically incorporated by reference herein for the material containedin them that is discussed in the sentence in which the reference isrelied upon.

Definitions

In this specification and in the claims that follow, reference will bemade to a number of terms, which shall be defined, to have the followingmeanings:

Throughout the specification and claims the word “comprise” and otherforms of the word, such as “comprising” and “comprises,” means includingbut not limited to, and is not intended to exclude, for example, otheradditives, components, integers, or steps.

As used in the description and the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “an enzyme” includesmixtures of two or more such enzymes, reference to “the probiotic”includes mixtures of two or more such probiotics, and the like.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. “About” can mean within 5%of the stated value. When such a range is expressed, another aspectincludes from the one particular value and/or to the other particularvalue. Similarly, when values are expressed as approximations, by use ofthe antecedent “about,” it will be understood that the particular valueforms another aspect. It will be further understood that the endpointsof each of the ranges are significant both in relation to the otherendpoint, and independently of the other endpoint. It is also understoodthat there are a number of values disclosed herein, and that each valueis also herein disclosed as “about” that particular value in addition tothe value itself. For example, if the value “5” is disclosed, then“about 5” is also disclosed.

Disclosed herein are fragments or variants of polypeptides, and anycombination thereof. The term “fragment” or “variant” when referring topolypeptide binding domains or binding molecules of the presentinvention include any polypeptides which retain at least some of theproperties (e.g., coagulation activity for an FVIII variant or fragment,or FVIII binding activity for the vWF fragment, or recycling activity byan albumin fragment) of the reference polypeptide. Fragments ofpolypeptides include proteolytic fragments, as well as deletionfragments, in addition to specific antibody fragments discussedelsewhere herein, but do not include the naturally occurring full-lengthpolypeptide (or mature polypeptide). Variants of polypeptide bindingdomains or binding molecules of the present invention include fragmentsas described above, and also polypeptides with altered amino acidsequences due to amino acid substitutions, deletions, or insertions.Variants can be naturally or non-naturally occurring. Non-naturallyoccurring variants can be produced using art-known mutagenesistechniques. Variant polypeptides can comprise conservative ornon-conservative amino acid substitutions, deletions or additions.

“Von Willebrand Factor,” also referred to herein as “vWF,” is a bloodglycoprotein involved in hemostasis. The basic vWF monomer is a2050-amino acid protein. Every monomer contains a number of specificdomains with a specific function, including the D′D3 domain, which bindsto factor VIII (Von Willebrand factor type D domain).

The term “endogenous vWF” as used herein indicates vWF moleculesnaturally present in plasma. The endogenous vWF molecule can bemultimer, but can also be a monomer or a dimer. Endogenous vWF in plasmabinds to FVIII and forms a non-covalent complex with FVIII.

The term “albumin fragment” as used herein means any albumin fragment orvariant of full-length albumin that retains the ability to prolong thehalf-life of the fusion protein, as described herein. Such albuminfragments and variants are known to those of skill in the art. Forexample, Otagiri et al (2009), Biol. Pharm, Bull. 32(4), 527-534,discloses that 77 albumin variants are known, of these 25 have mutationsin domain III. A natural variant lacking the C-terminal 175 amino acidsat the carboxy terminus has been shown to have a reduced half-life(Andersen et al (2010), Clinical Biochemistry 43, 367-372). Iwao et al(2007) studied the half-life of naturally occurring human albuminvariants using a mouse model, and found that K541 E and K560E hadreduced half-life, E501 K and E570K had increased half-life and K573Ehad almost no effect on half-life (Iwao, et al (2007) B.B.A. Proteinsand Proteomics 1774, 1582-1590). Galliano et al (1993) Biochim. Biophys.Acta 1225, 27-32 discloses a natural variant E505K. Minchiotti et al(1990) discloses a natural variant K536E. Minchiotti et al (1987)Biochim. Biophys. Acta 916, 41 1-418 discloses a natural variant K574N.Takahashi et al (1987) Proc. Natl. Acad. Sci. USA 84, 4413-4417,discloses a natural variant D550G. Carlson et al (1992). Proc. Nat.Acad. Sci. USA 89, 8225-8229, discloses a natural variant D550A. Theseare all incorporated by reference in their entirety for their teachingsconcerning albumin fragments and variants.

The term “vWF fragment” or “vWF fragments” used herein means any vWFfragments that interact with FVIII and retain at least one or moreproperties that are normally provided to FVIII by full-length vWF, e.g.,preventing premature activation to FVIIIa, preventing prematureproteolysis, preventing association with phospholipid membranes thatcould lead to premature clearance, preventing binding to FVIII clearancereceptors that can bind naked FVIII but not vWF-bound FVIII, and/orstabilizing the FVIII heavy chain and light chain interactions. The term“vWF fragment” as used herein does not include full length-or mature vWFprotein. In a particular embodiment, the “vWF fragment” as used hereincomprises a D′ domain and a D3 domain of the VWF protein, but does notinclude the A1 domain, the A2 domain, the A3 domain, the D4 domain, theB1 domain, the B2 domain, the B3 domain, the C1 domain, the C2 domain,and the CK domain of the vWF protein. vWF fragments and variants areknown to those of skill in the art and are disclosed herein.

A “fusion” or “chimeric” protein comprises a first amino acid sequencelinked to a second amino acid sequence with which it is not naturallylinked in nature. In one embodiment, the term “fusion protein,” as usedherein, in one example refers to the fusion of the von Willebrand'sfactor fragment, e.g. D′D3 to albumin, or an albumin fragment. Alsodisclosed is albumin linked to Tissue Factor (TF) for Factor VIIa.

Disclosed herein is a “modifying molecule.” A modifying molecule is anymolecule capable of modifying a coagulation factor so that it mayinteract with a fusion protein while retaining the coagulation enhancingactivity of the coagulation factor, e.g. factor VIII (FVIII). Forexample, a FVIII can be modified with a polyethylene glycol chain andcapped by a fatty acid. Various examples of modifying molecules arediscussed herein.

A “modified coagulation factor” refers to a coagulation factor which hasbeen modified by a modifying molecule so that it is capable ofinteracting with a fusion protein while retaining sufficient coagulationactivity. The modified coagulation factor can also be referred to as aderivatized FVIII or F8F herein. The modified coagulation factor can,for example, be bound to a fusion protein of D′D3 attached by anappropriate sized linker to human albumin in such a way that albumin canbind the fatty acid attached to the modified FVIII to form the modifiedcoagulation factor complex, for example the Factor VIII or Factor VIIacomplex of the subject invention.

As used herein, the term “half-life” refers to a biological half-life ofa particular polypeptide in vivo. Half-life may be represented by thetime required for half the quantity administered to a subject to becleared from the circulation and/or other tissues in the animal.

The term “half-life limiting factor” or “FVIII half-life limitingfactor” as used herein indicates a factor that prevents the half-life ofa FVIII protein from being longer than 1.5 fold or 2 fold compared towild-type FVIII. For example, full length or mature vWF can act as aFVIII half-life limiting factor by inducing the FVIII and vWF complex tobe cleared from the system by one or more vWF clearance pathways. In oneexample, endogenous vWF is a FVIII half-life limiting factor. In anotherexample, a full-length recombinant vWF molecule non-covalently bound toa FVIII protein can be a FVIII-half-life limiting factor.

The terms “interacts with” or “linked to” as used herein refers in oneembodiment to a covalent or non-covalent linkage. The term “covalentlylinked” or “covalent linkage” refers, for example, to a covalent bond,e.g., a disulfide bond, a peptide bond, or one or more amino acids. Inanother embodiment “interacts with” or “linked to” means the proteins orprotein fragments disclosed herein are connected by a linker between thetwo proteins or protein fragments that are linked together, for example,between the FVIII and the albumin, and/or between the D′D3 and albumin.The first amino acid can be directly joined or juxtaposed to the secondamino acid or alternatively an intervening sequence can covalently jointhe first sequence to the second sequence. The term “linked” can meannot only a fusion of a first amino acid sequence to a second amino acidsequence at the C-terminus or the N-terminus, but also includesinsertion of the whole first amino acid sequence (or the second aminoacid sequence) into any two amino acids in the second amino acidsequence (or the first amino acid sequence, respectively). In oneembodiment, the first amino acid sequence can be joined to a secondamino acid sequence by a peptide bond or a linker. The linker can be apeptide or a polypeptide or any chemical moiety, for example clickchemistry.

The coagulation factor complexes disclosed herein can be usedprophylactically. As used herein the term “prophylactic treatment”refers to the administration of a molecule prior to a bleeding episodeor consistently during normal activity to prevent a bleeding episode. Inone embodiment, the subject in need of a general hemostatic agent isundergoing, or is about to undergo, surgery. The coagulation factorcomplex can be administered prior to or after surgery as a prophylactic.The coagulation factor complex can be administered during or aftersurgery to control an acute bleeding episode. The surgery can include,but is not limited to, liver transplantation, liver resection, dentalprocedures, or stem cell transplantation.

The coagulation factor complexes of the invention can also be used foron-demand (also referred to as “episodic”) treatment. The term“on-demand treatment” or “episodic treatment” refers to theadministration of a chimeric molecule in response to symptoms of ableeding episode or before an activity that may cause bleeding. In oneaspect, the on-demand (episodic) treatment can be given to a subjectwhen bleeding starts, such as after an injury, or when bleeding isexpected, such as before surgery. In another aspect, the on-demandtreatment can be given prior to activities that increase the risk ofbleeding, such as contact sports.

As used herein the term “acute bleeding” refers to a bleeding episoderegardless of the underlying cause. For example, a subject may havetrauma, uremia, a hereditary bleeding disorder (e.g., factor VIIdeficiency) a platelet disorder, or resistance owing to the developmentof antibodies to clotting factors.

Treat, treatment, treating, as used herein refers to, e.g., thereduction in severity of a disease or condition; the reduction in theduration of a disease course; the amelioration of one or more symptomsassociated with a disease or condition; the provision of beneficialeffects to a subject with a disease or condition, without necessarilycuring the disease or condition, or the prophylaxis of one or moresymptoms associated, with a disease or condition.

In one embodiment, the term “treating” or “treatment” means maintaininga FVIII trough level at least about 1 IU/dL, 2 IU/dL, 3 IU/dL, 4 IU/dL,5 IU/dL, 6 IU/dL, 7 IU/dL, 8 IU/dL, 9 IU/dL, 10 IU/dL, 11 IU/dL, 12IU/dL, 13 IU/dL, 14 IU/dL, 15 IU/dL, 16 IU/dL, 17 IU/dL, 18 IU/dL, 19IU/dL, or 20 IU/dL, in a subject by administering a coagulation factorcomplex of the invention. In another embodiment, treating or treatmentmeans maintaining a FVIII trough level between about 1 and about 20IU/dL, about 2 and about 20 IU/dL, about 3 and about 20 IU/dL, about 4and about 20 IU/dL, about 5 and about 20 IU/dL, about 6 and about 20IU/dL, about 7 and about 20 IU/dL, about 8 and about 20 IU/dL, about 9and about 20 IU/dL, or about 10 and about 20 IU/dL. Treatment ortreating of a disease or condition can also include maintaining FVIIIactivity in a subject at a level comparable to at least about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, or 20% of the FVIII activity in a non-hemophiliac subject. Theminimum trough level required for treatment can be measured by one ormore known methods and can be adjusted (increased or decreased) for eachperson.

Coagulation Factor Complexes

Human albumin has a series of properties that are useful in theconstruction of fusion proteins described herein. It has the ability toprolong the half life of fusion proteins by binding to the neonatal Fcreceptor and it binds a number of small molecules including fatty acidsand bilirubin. In addition, it has a single exposed sulfhydryl groupthat can be utilized to attach various ligands. As utilized herein theseproperties allow the construction of a long half life FVIII, as well asa long half life, high activity version of FVIIa. The coagulation factorcomplexes disclosed herein comprise: a coagulation factor; a fusionprotein comprising a first protein fused to albumin, or an albuminfragment; and a modifying molecule, wherein the modifying molecule iscoupled to the coagulation factor in such a way as to allow binding bythe fusion protein, thereby creating a modified coagulation factor;wherein the modified coagulation factor and the fusion protein interactin at least two independent sites. The modifying molecule can also becoupled to the fusion protein in such a way as to allow binding of thefusion protein to the coagulation factor or to cause a chemical reactionbetween the coagulation factor and the fusion protein. The combinationof the coagulation factor with the modifying molecule can be referred toa modified coagulation factor, e.g., modified Factor VIII and VIIa,herein. The modified coagulation factor and the fusion protein interactin at least two independent sites. The coagulation factor can be aFactor VIIa, for example. In another embodiment, the coagulation factorcan be Factor VIII.

Coagulation Factor

vWF is a very large molecule and circulates as a large multimericcomplex of these large molecules (Leming, Christophe, & Denis, 2015). Itis so large that it is ingested into macrophages and digested as aparticle. Attempts to engineer a smaller fragment of vWF, called D′D3,that protects FVIII have been successful and fusing this fragment to theFc region increases its half life dramatically (Yee et al., 2014), InvWF deficient mice, this D′D3-Fc fusion increases the half life of FVIIIfrom about 15 hours to over 7 days.

The binding constant for vWF and FVIII is about 0.3 nM (Orlova, Kovnir,Vorohiev, Gabibov, & Vorobiev, 2013). The measured binding constant ofthe D′D3-Fc fusion created by Yee, et al. (Yee et al., 2014) is 1.5 nM.FVIII binds tightly but reversibly to vWF such that there is alwaysabout 1 to 2 percent of the FVIII free in solution. In both mice andhumans, vWF exists at about a filty-fold higher concentration thanFVIII. Between the lower binding constant. and the much higherconcentration of vWF, FVIII can be quickly competed away from theD′D3-Fc fusion.

One potential solution to these problems is found by fusing D′D3 toalbumin, thereby creating a “fusion protein,” as it is referred toherein. Albumin is the most abundant protein in the blood (Peters,1995). Its 19 day half life in the circulation is determined by itsability to bind to the FcRN, as described earlier. It serves two majorroles: one is to maintain the osmolarity of the blood and the second isto transport hydrophobic molecules. Albumin is the major transporter offatty acids. A strategy that has been employed successfully to increasethe half life of insulin, for example, is to conjugate insulin tomyristic acid, a 14 carbon fatty acid. This molecule is called insulindetemir (Philips & Scheen, 2006). When injected, the fatty acid quicklybinds to albumin, increasing the half life of the insulin from 4 minutesto 5 hours.

A combination of these two ideas is disclosed herein (FIG. 1) in a novelmolecule to solve the long existing half life problem and provide a muchneeded solution. For example, first, the FVIII can be modified with apolyethylene glycol chain and capped by a fatty acid, for example. Thisexample of a “modifying molecule,” as it is referred to herein, createsan example of a “modified coagulation factor.” The fatty acid thusprotrudes from the FVIII molecule in one embodiment to create themodified coagulation factor. The modified coagulation factor can also bereferred to as a derivatized FVIII or F8F herein. Next, the modifiedcoagulation factor can be bound to a fusion protein of D′D3, derivedfrom vWF, attached by an appropriate sized linker to human albumin, insuch a way that albumin can bind the fatty acid attached to the modifiedFVIII to form the modified coagulation factor complex, for example theFactor VIII or Factor VIIa complex of the subject invention. The D′D3fragment can bind to its cognate site on the modified FVIII and thefatty acid can bind to albumin. The modified coagulation factor can betethered to the fusion protein at two points, both of which have strongbinding constants. In addition, the length of the linker connecting D′D3to albumin in the fusion protein can be altered, either longer orshorter, such that D′D3, can be properly or optimally orientated withthe Factor VIII binding site for optimal half life extension. In thisway, it is much less likely that native vWF will be able to competeeffectively for the modified FVIII and attachment to albumin increasesthe half life substantially.

The modified coagulation factor and the fusion protein can interact atone, two, three, four, or more sites. In one embodiment, the modifiedcoagulation factor and the fusion protein interact at two independentsites on both molecules. By “independent sites” is meantnon-overlapping, or distinct, areas of one, or both, molecules. At leastone binding site of the modified coagulation factor can be a naturalbinding site. In other words, the binding site is naturally occurring onthe coagulation factor, and is not part of its modification. The otherbinding site on the modified coagulation factor can be modified, suchthat one or more amino acids in that site is not natural, or native, tothe coagulation factor.

The fusion protein can comprise two, three, four, or more proteins fusedtogether. For example, the first fusion protein can comprise a D′D3fragment of von Willebrand's factor. Variants and fragments of vWF areknown to those of skill in the art, and are contemplated herein.Examples of such can be found in U.S. Pat. No. 9,125,890, and U.S.Patent Applications 2014/0357564 and 2013/120939. Alternatively, thefirst fusion protein can comprise Tissue Factor (TF). The second proteincan comprise albumin, or an immunoglobulin Fc fragment. In one example,the immunoglobulin Fc fragment can comprise a single chain variableregion (scFv) specific to the modified coagulation factor. The scFv canbe specific to a modified site of the modified coagulation factor.

FIG. 5 demonstrates that albumin can bind to the modified FVIII and thatit again has no effect on FVIII activity. Panel A shows chromatographyof FVIII modified with maleimide-PEG1000-laurate on a Superdex 200 sizeexclusion column, assayed by FVIII activity. Panel B shows that theaddition of albumin to the derivatized FVIII shifts the molecular weighthigher, as expected. Panel C demonstrates that albumin bound toderivatized FVIII does not compromise FVIII activity.

Two versions of the fusion protein have been produced by transfectioninto C3A cells (ATCC CRL-10741), a human liver cell line. CM110 in FIG.6, panel A, consists of the D′D3 fragment of von Willebrand Factor, a 56amino acid glycine, serine rich linker and a full length human albumin.CM210 (FIG. 6, panel B) is a dimer of this molecule, joined bysulfhydryl links through the D′D3 region. When FVIII modified witheither maleimide-PEG1000-laurate or maleimide-PEG1000-myristate isincubated with CM 110, they spontaneously form the modified coagulationfactor complex that shifts the activity of the FVIII to a highermolecular weight, as shown in FIG. 6, Panel C. While CM110 was in excessin this experiment, it is important to note that all of the FVIIIactivity is shifted to the higher molecular weight. Neither CM110 norCM210 affect activity of modified FVIII (FIG. 6, Panel D).

This dual binding strategy can be accomplished in a number of other wayswhile still maintaining the required FcRN cycling, as those of skill inthe art will appreciate in view of this disclosure. Other ligands canreplace the fatty acid in the modifying molecule, since albumin is knownto bind a wide variety of ligands, such as bilirubin (Peters, 1995).

Another embodiment is to substitute an antibody/small molecule set forthe albumin/fatty acid pair. There are many small molecules that havecognate monoclonal antibodies and these are often used for detection ofthe small molecule in biological specimens (Bradbury, Sidhu, Dübel, &McCafferty, 2011). A molecule can be constructed that has D′D3, an aminoacid spacer, the Fc region of the immunoglobulins and a single chainvariable region, specific for a small molecule, for example,nitrotyrosine. The modifying molecule could then take the form ofmaleimide-PEG1000-nitrotyrosine. This would have the same dual bindingeffect and FcRN cycling, but using immunoglobulin based m

Another alternative using a similar strategy of modifying FVIII can beused to create a covalent link between the coagulation factor and thefusion protein. Click chemistry or bio-orthogonal chemistry describesmolecules that are designed to react only with one another in a complexchemical milieu. Panel A in FIG. 7 shows two such molecules of thegeneral structure maleimide-PEG_(N)-X where in this case X is methyltetrazine on one and trans cyclooctene (TCO) on the other. As before,modifying FVIII with the methyl tetrazine containing molecule has noeffect on activity (Panel B, FIG. 7). The only free sulfhydryls on CMI10and CM210, the fusion proteins described herein, are those correspondingto cysteine 34 in albumin. By modifying CM110 or CM210 withmalehnide-PEG13-TCO and FVIII with maleimide-PEG4-methyltetrazine, thenmixing, a covalently linked complex forms when TCO reacts withmethyltetrazine. Panel C shows that addition of maleimide-PEG3-TCOmodified CM110 to maleimide-PEG4-methyl tetrazine modified FVIII resultsin a high molecular weight complex that still retains the FVIIIactivity. Panel D again demonstrates that formation of the covalentcomplex does not affect activity.

The mathematics of intramolecular binding has been described by Kramerand Karpen (Kramer & Karpen, 1998) and in more detail by Zhou (Zhou,2006). Binding becomes a function of the individual dissociationconstants and the effective local concentration. The bond betweenalbumin in the fusion protein and the modified FVIII, for example,tethers the D′D3 fragment to the modified FVIII. The local concentrationof the D′D3 fragment then becomes quite high, precluding binding ofendogenous vWF. Since the dissociation constants for either D′D3 bindingto FVIII or fatty acid binding to albumin are in the nanomolar range,binding of the fusion protein to modified FVIII should be tight. In thecase of the click chemistry modified complex, the link is covalent.

This dual binding strategy overcomes at least two problems encounteredby Yee, et al. ²(Yee et al., 2014). The first is to increase the bindingaffinity of FVIII for the fusion protein and prevent dilution by theexisting high concentration of vWF in the serum. Binding should now be aproduct of both binding constants and the effective concentration soneither free vWF nor free fatty acids should be able to competeeffectively for binding.

This molecule, referred to herein as the modified coagulation factorcomplex, has several desirable features not afforded by either FVIII orother long half life FVIII molecules. First, the very tight or covalentbinding ensures that there is very little dissociation of the modifiedFVIII from the fusion protein, preventing loss of the administered FVIIIinto the large pool of normal vWF. Second, by divorcing the modifiedcoagulation factor complex from the endogenous vWF and using the fusionprotein to extend the half life, it should obtain a half-life similar tothat shown by Yee, et al. (Yee et al., 2014) for their D′D3-Fc protein,which was over seven days. Third, by administering the modifiedcoagulation complex rather than free FVIII, it can reduce the incidenceof inhibitor formation. Fourth, by attaching the albumin to the fusionprotein, rather than directly to the FVIII, the FVIII activity ispreserved. Direct fusion of albumin to FVIII results in an inactivemolecule {Powell:2015gu}. Positioning albumin away from direct contactwith the FVIII should assist efficient recycling by the FcRN. Finally,this is an entirely human protein produced in a human cell system, whichcan further reduce the incidence of inhibitor formation.

The half-life of the coagulation factor complex comprising the modifiedcoagulation factor VIII can be at least 10, 20, 30, 40, 50, 60, 70, 80,90, or 100% or greater compared to a coagulation factor alone. Thecoagulation factor complex can also have a half-life that is 2, 3, 4, 5,6, 7, 8, 9, or 10 or more times longer, when compared to an unmodifiedcoagulation factor. More specifically, the half-life of the coagulationfactor complex can be at least about 1.5 times, at least about 2 times,at least about 2.5 times, at least about 3 times, at least about 4times, at least about 5 times, at least about 6 times, at least about 7times, at least about 8 times, at least about 9 times, at least about 10times, at least about 11 times, or at least about 12 times longer ormore than the half-life of a FVIII protein alone. In one embodiment, thehalf-life of FVIII is about 1.5-fold to about 20-fold, about 1.5 fold toabout 15 fold, or about 1.5 fold to about 10 fold longer than thehalf-life of wild-type FVIII. In another embodiment, the half-life ofthe FVIII when in the coagulation factor complex is extended about2-fold to about 10-fold, about 2-fold to about 9-fold, about 2-fold toabout 8-fold, about 2-fold to about 7-fold, about 2-fold to about6-fold, about 2-fold to about 5-fold, about 2-fold to about 4-fold,about 2-fold to about 3-fold, about 2.5-fold to about 10-fold, about2.5-fold to about 9-fold, about 2.5-fold to about 8-fold, about 2.5-foldto about 7-fold, about 2.5-fold to about 6-fold, about 2.5-fold to about5-fold, about 2.5-fold to about 4-fold, about 2.5-fold to about 3-fold,about 3-fold to about 10-fold, about 3-fold to about 9-fold, about3-fold to about 8-fold, about 3-fold to about 7-fold, about 3-fold toabout 6-fold, about 3-fold to about 5-fold, about 3-fold to about4-fold, about 4-fold to about 6fold, about 5-fold to about 7-fold, orabout 6-fold to about 8 fold as compared to wild-type FVIII or a FVIIIprotein alone. In other embodiments, the half-life of the coagulationfactor complex is at least about 17 hours, at least about 18 hours, atleast about to 19 hours, at least about 20 hours, at least about 21hours, at least about 22 hours, at least about 23 hours, at least about24 hours, at least about 25 hours, at least about 26 hours, at leastabout 27 hours, at least about 28 hours, at least about 29 hours, atleast about 30 hours, at least about 31 hours, at least about 32 hours,at least about 33 hours, at least about 34 hours, at least about 35hours, at least about 36 hours, at least about 48 hours, at least about60 hours, at least about 72 hours, at least about 84 hours, at leastabout 96 hours, or at least about 108 hours. In still other embodiments,the half-life of the coagulation factor complex is about 15 hours toabout two weeks, about 16 hours to about one week, about 17 hours toabout one week, about 18 hours to about one week, about 19 hours toabout one week, about 20 hours to about one week, about 21 hours toabout one week, about 22 hours to about one week, about 23 hours toabout one week, about 24 hours to about one week, about 36 hours toabout one week, about 48 hours to about one week, about 60 hours toabout one week, about 24 hours to about six days, about 24 hours toabout five days, about 24 hours to about four days, about 24 hours toabout three days, or about 24 hours to about two days.

In some embodiments, the average half-life of the coagulation factorcomplex per subject is about 15 hours, about 16 hours, about 17 hours,about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22hours, about 23 hours, about 24 hours (1 day), about 25 hours, about 26hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours,about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35hours, about 36 hours, about 40 hours, about 44 hours, about 48 hours (2days), about 54 hours, about 60 hours, about 72 hours (3 days), about 84hours, about 96 hours (4 days), about 108 hours, about 120 hours (5days), about six days, about seven days (one week), about eight days,about nine days, about 10 days, about 11 days, about 12 days, about 13days, or about 14 days.

Long Half Life, High Activity FVIIa

A similar strategy can be applied to creating a long half-life, highactivity FVIIa (FIG. 3). Under normal coagulation conditions, FVII isfirst activated by proteolysis to FVIIa. FVIIa, however has very lowactivity for carrying out its function, which to activate Factor X (FX).When FVIIa encounters membrane bound Tissue Factor at the site ofinjury, it binds to form a complex with over 100 fold higher activity inproduction of FXa (Vadivel & Bajaj, 2012). Ordinarily, FVIIa is presentat very low concentrations and only serves to set off the amplifyingcascade of blood coagulation. As a bypass agent, FVIIa is administeredat very high concentrations such that it can activate sufficient FX toFXa to provide the thrombin burst required to form a fibrin clot.

Similar to the long half life FVIII example disclosed above, TF has adissociation constant for FVIIa of about 1 nM (Vadivel & Bajaj, 2012).By first derivatizing FVIIa with a water soluble fatty acid, thenbinding it to a soluble Tissue Factor—albumin fusion protein, a complexwith several desirable properties should be created. The complex canhave 2, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more foldincreased activity with respect to activation of FX. This allowseffective bypass therapy with substantially less protein than iscurrently used, reducing both cost and the chance of immunogenicity. Thehalf life of the complex can be increased by virtue of recycling viaFcRN as a result of the albumin fusion, resulting in less frequentadministration. This is an important consideration for FVIIa due to itsvery short 2 hour half-life. Even a modest extension is helpful, butthis molecule can confer a particularly long half life. Binding ofantithrombin III (ATIII) to FVIIa is one of the main routes of clearanceof FVIIa activity (Lawson, Butenas, Ribarik, & Mann, 1993). ATIII ismost effective in inactivating FVIIa in the presence of heparin and whenTF is membrane bound. Studies of ATIII mediated inactivation of FVIIahave demonstrated that ATIII displaces FVIIa from TF and then preventsrebinding. The presence of a tightly attached TF should prevent bindingof the derivatized FVIIaF to membrane bound TF and perhaps prevent ATIIIinactivation.

Modifying Molecules

“Modifying molecules,” as disclosed herein, can comprise any moleculewhich modifies a coagulation factor and renders it capable ofinteracting with a fusion protein. The modifying molecule can, forexample, comprise a fatty acid. The modifying molecule can be attachedto the modified coagulation factor through a polyethylene glycol chain,for example. A first and a second protein of the fusion protein can bejoined together via a linker, for example. The modified coagulationfactor can comprise one or more modified amino acids. Additionally, oralternatively, the fusion protein can comprise modified amino acids. Forexample, coagulation factor complexes of the invention can, in someembodiments, be composed of amino acids joined to each other by peptidebonds or modified peptide bonds, i.e., peptide isosteres, and maycontain amino acids other than the 20 gene-encoded amino acids. Thepolypeptides may be modified by either natural processes, such aspost-translational processing, or by chemical modification techniqueswhich are well known in the art. Such modifications are well describedin basic texts and in more detailed monographs, as well as in avoluminous research literature.

Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic polypeptides may result fromposttranslation natural processes or may be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, (GPI anchor formation,hydroxylation, iodination, methylation, myristylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination.(See, for instance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993);POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson,Ed., Academic Press, New York; pgs. 1-12 (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci.663:48-62 (1992)).

FVIII can be modified by a number of modifiying molecules of the generalstructure maleimide-PEGn-X, shown in Table 1. A variety of suchmodifying molecules can be used. For example, various modifications ofmaleimide-PEG are known to those of skill in the art, such as thosepresented in U.S. Pat. No. 6,828,401, hereby incorporated by referencein its entirety for its disclosure concerning PEG-maleimide derivatives.In the examples of this invention, this modification did not have anyobserved effect on the activity of FVIII. FIG. 3 shows FVIII activitybefore and after modification with either Mal-PEG-laurate orMal-PEG-FITC. FIG. 4 shows SDS containing polyacrylamide gels. Panel Ais a silver stained gel showing that derivatization has no effect on themolecular weight or structure of FVIII. Panel B shows a protein blot ofanother gel that was then probed with streptavidin-HRP to visualize themodifying molecule, in this case, maleimide-PEG5000-biotin. The resultshows that the Mal-PEG-biotin molecule reacts with the heavy chain ofFVIII. Examples of modifying molecules can be found in Table 1.

TABLE 1. Molecules used to modify FVIII

-   -   Maleimide-PEG5000-biotin    -   Maleimide-PEG1000-fluorescein isothiocyanate    -   Maleimide-PEG1000-laurate    -   Maleimide-PEG1000-myristate    -   Maleimide-PEG4-6-methyl tetrazine    -   Maleimide-PEG3-trans cyclooctene

The chemical moieties for modification of the coagulation factor may beselected from water soluble polymers such as polyethylene glycol,ethylene glycol/propylene glycol copolymers, carboxymethylcellulose,dextran, polyvinyl alcohol and the like. The coagulation factors may bemodified at random positions within the molecule, or at predeterminedpositions within the molecule and may include one, two, three or moreattached chemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog). For example,the polyethylene glycol may have an average molecular weight of about200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000,70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

As noted above, the polyethylene glycol may have a branched structure.Branched polyethylene glycols are described, for example, in U.S. Pat.No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72(1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999);and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosuresof each of which are incorporated herein by reference.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, such as, for example, themethod disclosed in EP 0 401 384 (coupling PEG to G-CSF), hereinincorporated by reference; see also Malik et al., Exp. Hematol,20:1028-1035 (1992), reporting pegylation of GM-CSF using tresylchloride. For example, polyethylene glycol may be covalently boundthrough amino acid residues via reactive group, such as a free amino orcarboxyl group. Reactive groups are those to which an activatedpolyethylene glycol molecule may be bound. The amino acid residueshaving a free amino group may include lysine residues and the N-terminalamino acid residues; those having a free carboxyl group may includeaspartic acid residues glutamic acid residues and the C-terminal aminoacid residue. Sulthydryl groups may also be used as a reactive group forattaching the polyethylene glycol molecules. Preferred for therapeuticpurposes is attachment at an amino group, such as attachment at theN-terminus or lysine group.

As suggested above, polyethylene glycol may be attached to proteins vialinkage to any of a number of amino acid residues. For example,polyethylene glycol can be linked to proteins via covalent bonds tolysine, histidine, aspartic acid, glutamic acid, or cysteine residues.One or more reaction chemistries may be employed to attach polyethyleneglycol to specific amino acid residues (e.g., lysine, histidine,aspartic acid, glutamic acid, or cysteine) of the protein or to morethan one type of amino acid residue (e.g., lysine, histidine, asparticacid, glutamic acid, cysteine and combinations thereof) of the protein.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (polypeptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

As indicated above, pegylation of the coagulation factors of theinvention may be accomplished by any number of means. For example,polyethylene glycol may be attached to the molecule either directly orby an intervening linker. Linkerless systems for attaching polyethyleneglycol to proteins are described in Delgado et al., Crit. Rev. Thera.Drug Carrier Sys. 9:249-304 (1992); Francis et al., Intern. J. ofHematol. 68:1-18 (1998); U.S. Pat. Nos. 4,002,531; 5,349,052; WO95/06058; and WO 98/32466, the disclosures of each of which areincorporated herein by reference.

One system for attaching polyethylene glycol directly to amino acidresidues of proteins without an intervening linker employs tresylatedMPEG, which is produced by the modification of monmethoxy polyethyleneglycol (MPEG) using tresylchloride. Upon reaction of protein withtresylated MPEG, polyethylene glycol is directly attached to aminegroups of the protein. Thus, the invention includes protein-polyethyleneglycol conjugates produced by reacting proteins of the invention with apolyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonylgroup.

Polyethylene glycol can also be attached to proteins using a number ofdifferent intervening linkers. For example, U.S. Pat. No. 5,612,460, theentire disclosure of which is incorporated herein by reference,discloses urethane linkers for connecting polyethylene glycol toproteins. Protein-polyethylene glycol conjugates wherein thepolyethylene glycol is attached to the protein by a linker can also beproduced by reaction of proteins with compounds such asMPEG-succinimidylsuccinate, MPEG activated with1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate,MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. Anumber of additional polyethylene glycol derivatives and reactionchemistries for attaching polyethylene glycol to proteins are describedin International Publication No. WO 98/32466, the entire disclosure ofwhich is incorporated herein by reference. Pegylated protein productsproduced using the reaction chemistries set out herein are includedwithin the scope of the invention.

The number of polyethylene glycol moieties attached to a modifiedcoagulation factor of the invention (i.e., the degree of substitution)may also vary. For example, the pegylated proteins of the invention maybe linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20,or more polyethylene glycol molecules. Similarly, the average degree ofsubstitution within ranges such as 1-3,2-4, 3-5,4-6, 5-7,6-8, 7-9,8-10,9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20polyethylene glycol moieties per protein molecule. Methods fordetermining the degree of substitution are discussed, for example, inDelgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

The polypeptides of the invention can be recovered and purified fromchemical synthesis and recombinant cell cultures by standard methodswhich include, but are not limited to, ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification. Well known techniques forrefolding protein may be employed to regenerate active conformation whenthe polypeptide is denatured during isolation and/or purification.

The presence and quantity of modified coagulation factor complexes ofthe invention may be determined using ELISA, a well known immunoassayknown in the art. In one ELISA protocol that would be useful fordetecting/quantifying modified molecules of the invention, comprises thesteps of coating an ELISA plate with an anti-human serum albuminantibody, blocking the plate to prevent non-specific binding, washingthe ELISA plate, adding a solution containing the molecule of theinvention (at one or more different concentrations), adding a secondaryanti-therapeutic protein specific antibody coupled to a detectable label(as described herein or otherwise known in the art), and detecting thepresence of the secondary antibody. In an alternate version of thisprotocol, the ELISA plate might be coated with the anti-therapeuticprotein specific antibody and the labeled secondary reagent might be theanti-human albumin superfamily specific antibody.

Polypeptide and Polynucleotide Fragments and Variants

The present invention is further directed to fragments of thecoagulation factor complexes described herein as well as fragments ofindividual components of the coagulation factor complexes, such as themodified coagulation factor, the modifying molecule, or the fusionprotein. These modifications can include those disclosed herein, whichmodify the molecules in such a way as to increase activity or half life,or other modifications that enhance the properties of the molecule ormake it desirable for other reasons.

Even if a deletion of one or more amino acids results in modificationsor loss of one or more functions, the coagulation function of thecomplex may still be retained. Accordingly, fragments of the moleculesdisclosed herein, include the full length protein as well aspolypeptides having one or more residues deleted from the amino acidsequence of the reference polypeptide, are contemplated herein.

The present application is directed to proteins containing polypeptidesat least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99% identical to a reference polypeptide sequence (e.g., acoagulation factor, a modifying molecule, or a fusion protein) set forthherein, or fragments thereof.

“Variant” refers to a polynucleotide or nucleic acid differing front areference nucleic acid or polypeptide, but retaining essentialproperties thereof. Generally, variants are overall closely similar,and, in many regions, identical to the reference nucleic acid orpolypeptide.

As used herein, “variant” refers to a protein disclosed herein whichdiffers in sequence from the known sequence of the protein, but retainsat least one functional and/or therapeutic property thereof (e.g., atherapeutic activity and/or biological activity, including but notlimited to coagulation) as described elsewhere herein or otherwise knownin the art. Generally, variants are overall very similar, and, in manyregions, identical to the amino acid sequence of the protein of interestor albumin superfamily protein.

The present invention is also directed to proteins which comprise, oralternatively consist of, an amino acid sequence which is at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, for example,the amino acid sequence of the coagulation factor itself, the fusionprotein, or the modifying molecule. Fragments of these polypeptides arealso provided (e.g., those fragments described herein). Furtherpolypeptides encompassed by the invention are polypeptides encoded bypolynucleotides which hybridize to the complement of a nucleic acidmolecule encoding an amino acid sequence of the invention understringent hybridization conditions (e.g., hybridization to filter boundDNA in 6 times sodium chloride/sodium citrate (SSC) at about 45 degreesCelsius, followed by one or more washes in 0.2 times SSC, 0.1% SDS atabout 50-65 degrees Celsius), under highly stringent conditions (e.g.,hybridization to filter bound DNA in 6 times sodium chloride/sodiumcitrate (SSC) at about 45 degrees Celsius, followed by one or morewashes in 0.1 times SSC, 0.2% SDS at about 68 degrees Celsius), or underother stringent hybridization conditions which are known to those ofskill in the art (see, for example, Ausuhel, F. M. et al., eds., 1989Current protocol in Molecular Biology, Green publishing associates,Inc., and John Wiley & Sons Inc., New York, at pages 6.3.1-6.3.6 and2.10.3). Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% of the amino acid residues in thesubject sequence may be inserted, deleted, or substituted with anotheramino acid. These alterations of the reference sequence may occur at theamino- or carboxy-terminal positions of the reference amino acidsequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, theamino acid sequence of a coagulation factor or a fragment, can bedetermined conventionally using known computer programs. A preferredmethod for determining the best overall match between a query sequence(a sequence of the present invention) and a subject sequence, alsoreferred to as a global sequence alignment, can be determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237-245 (1990)). In a sequence alignment the query andsubject sequences are either both nucleotide sequences or both aminoacid sequences. The result of said global sequence alignment isexpressed as percent identity. Preferred parameters used in a FASTDBamino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1,Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, WindowSize=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, WindowSize=500 or the length of the subject amino acid sequence, whichever isshorter.

The polynucleotide variants of the invention may contain alterations inthe coding regions, non-coding regions, or both. Especially preferredare polynucleotide variants containing alterations which produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded polypeptide. Nucleotide variants producedby silent substitutions due to the degeneracy of the genetic code arepreferred. Moreover, polypeptide variants in which less than 50, lessthan 40, less than 30, less than 20, less than 10, or 5-50, 5-25, 5-10,1-5, or 1-2 amino acids are substituted, deleted, or added in anycombination are also preferred. Polynucleotide variants can be producedfor a variety of reasons, e.g., to optimize codon expression for aparticular host (change codons in the human mRNA to those preferred by abacterial host, such as, yeast or E. coli).

Naturally occurring variants are called “allelic variants,” and refer toone of several alternate forms of a gene occupying a given locus on achromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985)). These allelic variants can vary at either thepolynucleotide and/or polypeptide level and are included in the presentinvention. Alternatively, non-naturally occurring variants may beproduced by mutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the polypeptides of the present invention. Forinstance, one or more amino acids can be deleted from the N-terminus orC-terminus of the polypeptide of the present invention withoutsubstantial loss of biological function. As an example, Ron et al. (J.Biol. Chem. 268: 2984-2988 (1993)) reported variant KGF proteins havingheparin binding activity even after deleting 3, 8, or 27 amino-terminalamino acid residues. Similarly, Interferon gamma exhibited up to tentimes higher activity after deleting 8-10 amino acid residues from thecarboxy terminus of this protein, (Doheli et al., J. Biotechnology7:199-216 (1988).)

Thus, the invention further includes polypeptide variants which have afunctional activity (e.g., biological activity and/or therapeuticactivity). In highly preferred embodiments the invention providesmodifications to coagulation factors, which modifications allow for anincreased functional activity, such as a prolonged half-life.

Also disclosed are methods of treating a subject with a diseaserequiring coagulation factor infusion, the method comprisingadministering to the subject the coagulation factor complex disclosedherein. The disease can be hemophilia, for example. The administrationof the coagulation factor complex to the subject result can result in ablood level half-life of the coagulation factor complex which is greaterthan the blood level half-life obtained upon administration of thecoagulation factor alone. The coagulation factor complex can beadministered to the subject via injection, inhalation, internasally, ororally.

The modified coagulation factor complexes of the invention orformulations thereof may be administered by any conventional methodincluding parenteral (e.g. subcutaneous or intramuscular) injection orintravenous infusion. The treatment may consist of a single dose or aplurality of doses over a period of time.

The coagulation factor complexes disclosed herein can be present as apharmaceutical formulation, together with one or more acceptablecarriers. The carrier(s) must be “acceptable” in the sense of beingcompatible with the coagulation factor complex, and not deleterious tothe recipients thereof.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Such methods include the step of bringing into association thecoagulation factor complex with the carrier that constitutes one or moreaccessory ingredients. In general the formulations are prepared byuniformly and intimately bringing into association the active ingredientwith liquid carriers or finely divided solid carriers or both, and then,if necessary, shaping the product.

Kits

Also disclosed herein are kits comprising the coagulation factorcomplexes. Formulations or compositions of the invention may be packagedtogether with, or included in a kit with, instructions or a packageinsert referring to the extended shelf-life of the coagulation factorcomplex. For instance, such instructions or package inserts may addressrecommended storage conditions, such as time, temperature and light,taking into account the extended or prolonged shelf-life of thecoagulation factor complexes of the invention. Such instructions orpackage inserts may also address the particular advantages of thecoagulation factor complexes of the inventions, such as the ease ofstorage for formulations that may require use in the field, outside ofcontrolled hospital, clinic or office conditions. As described above,formulations of the invention may be in aqueous form and may be storedunder less than ideal circumstances without significant loss oftherapeutic activity.

Methods of Treating

The coagulation factor complexes and/or polynucleotides of the inventionmay be administered alone or in combination with other therapeuticagents. They may be administered in combination with other coagulationfactor complexes and/or polynucleotides of the invention. Combinationsmay be administered either concomitantly, e.g., as an admixture,separately but simultaneously or concurrently; or sequentially. Thisincludes presentations in which the combined agents are administeredtogether as a therapeutic mixture, and also procedures in which thecombined agents are administered separately but simultaneously, e.g., asthrough separate intravenous lines into the same individual.Administration “in combination” further includes the separateadministration of one of the compounds or agents given first, followedby the second.

In specific aspects, coagulation factor complex used in methods of thepresent invention can be contained in a formulation containing a buffer,a sugar and/or a sugar alcohol (including without limitation trehaloseand mannitol), a stabilizer (such as glycine), and a surfactant (such asPolysorbate 80). In further embodiments, the formulation may furtherinclude sodium, histidine, calcium, and glutathione.

In one aspect, the formulations comprising the coagulation factorcomplex are lyophilized prior to administration. Lyophilization iscarried out using techniques common in the art and should be optimizedfor the composition being developed (Tang et al., Pharm Res. 21 :191-200, (2004) and Chang et al, Pharin Res. 13:243-9 (1996).

Methods of preparing pharmaceutical formulations can include one or moreof the following steps: adding a stabilizing agent as described hereinto said mixture prior to lyophilizing, adding at least one agentselected from a hulking agent, an osmolality regulating agent, and asurfactant, each of which as described herein, to said mixture prior tolyophilization. A lyophilized formulation is, in one aspect, at leastcomprised of one or more of a buffer, a bulking agent, and a stabilizer.In this aspect, the utility of a surfactant is evaluated and selected incases where aggregation during the lyophilization step or duringreconstitution becomes an issue. An appropriate buffeting agent isincluded to maintain the formulation within stable zones of pH duringlyophilization.

The standard reconstitution practice for lyophilized material is to addback a volume of pure water or sterile water for injection (WFI)(typically equivalent to the volume removed during lyophilization),although dilute solutions of antibacterial agents are sometimes used inthe production of pharmaceuticals for parenteral administration (Chen,Drug Development and Industrial Pharmacy, 18: 131 1-1354 (1992)).

The lyophilized material may be reconstituted as an aqueous solution. Avariety of aqueous carriers, e,g., sterile water for injection, waterwith preservatives for multi dose use, or water with appropriate amountsof surfactants (for example, an aqueous suspension that contains theactive compound in admixture with excipients suitable for themanufacture of aqueous suspensions). In various aspects, such excipientsare suspending agents, for example and without limitation, sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia:dispersing or wetting agents are a naturally-occurring phosphatide, forexample and without limitation, lecithin, or condensation products of analkylene oxide with fatty acids, for example and without limitation,polyoxyethylene stearate, or condensation products of ethylene oxidewith long chain aliphatic alcohols, for example and without limitation,heptadecaethyl-eneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooieate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides, for example and without limitation, polyethylene sorbitanmonooieate. In various aspects, the aqueous suspensions also contain oneor more preservatives, for example and without limitation, ethyl, orn-propyl, p-hydroxybenzoate.

In certain embodiments, compositions of the present invention are liquidformulations for administration with the use of a syringe or otherstorage vessel. In further embodiments, these liquid formulations areproduced from lyophilized material described herein reconstituted as anaqueous solution. In a further aspect, the compositions of the inventionfurther comprise one or more pharmaceutically acceptable carriers. Thephrases “pharmaceutically” or “pharmacologically” acceptable refer tomolecular entities and compositions that are stable, inhibit proteindegradation such as aggregation and cleavage products, and in additiondo not produce allergic, or other adverse reactions when administeredusing routes well-known in the art, as described below.“Pharmaceutically acceptable carriers” include any and all clinicallyuseful solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like,including those agents disclosed above.

Single or multiple administrations of coagulation factor complexes arecarried out with the dose levels and pattern being selected by thetreating physician. For the prevention or treatment of disease, theappropriate dosage depends on the type of disease to be treated, theseverity and course of the disease, whether drug is administered forpreventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the drug, and the discretion of theattending physician.

In further embodiments and in accordance with any of the above,treatment of coagulation diseases such as Hemophilia A may involve aninitial treatment of coagulation factor complex alone or in combinationwith another agent, followed by one or more repeat doses of coagulationfactor complex and/or other agents. The nature of the initial and thenthe subsequent repeat administrations will depend in part on the diseasebeing treated.

In further aspects, coagulation factor complex can be administered to asubject in doses ranging from 0.5 IU/kg-200 IU kg. In some embodiments,coagulation factor complex is administered in doses ranging from 1-190,5-180, 10-170, 15-160, 20-450, 25-140, 30-130, 35-120, 40-110, 45-100,50-90, 55-80, or 60-70 IU/kg. In further embodiments and in accordancewith any of the above, coagulation factor complex can be administered toa subject at doses of between about 1 IU/kg to about 150 IU/kg. In stillfurther embodiments, the coagulation factor complex is administered atdoses of between 1.5 IU/kg to 150 IU/kg, 2 IU/kg to 50 IU/kg, 5 IU/kg to40 IU/kg, 10 IU/kg to 20 IU/kg, 10 IU/kg to 100 IU/kg, 25 IU/kg to 75IU/kg, and 40 IU kg to 75 IU/kg, In still further embodiments,coagulation factor complex is administered at 2, 5, 7.5, 10, 15, 20, 25,30, 35, 40, 45, or 50 IU/kg. As will be appreciated and as is discussedfurther herein, appropriate dosages of coagulation factor complex may beascertained through use of established assays for determining bloodlevel dosages in conjunction with appropriate dose-response data.

In certain examples, the complexes of the current invention can beinfused or admistered to the muscle to treat hemophilia A. Compositionsof coagulation factor complex can be contained in pharmaceuticalformulations, as described herein. Such formulations can be administeredorally, topically, transdermally, parenterally, by inhalation spray,vaginally, rectally, or by intracranial injection. The term parenteralas used herein includes subcutaneous injections, intravenous,intramuscular, intracisternal injection, or infusion techniques.Administration by intravenous, intradermal, intramuscular, intramammary,intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection andor surgical implantation at a particular site is contemplated as well.Generally, compositions are essentially free of pyrogens, as well asother impurities that could be harmful to the recipient.

In one aspect, formulations of the invention are administered by aninitial bolus followed by a continuous infusion to maintain therapeuticcirculating levels of drug product. As another example, the inventivecompound is administered as a one-time dose. Those of ordinary' skill inthe art will readily optimize effective dosages and administrationregimens as determined by good medical practice and the clinicalcondition of the individual patient. The route of administration can be,but is not limited to, by intravenous, intraperitoneal, subcutaneous, orintramuscular administration. The frequency of dosing depends on thepharmacokinetic parameters of the agents and the route ofadministration. The optimal pharmaceutical formulation is determined byone skilled in the art depending upon the route of administration anddesired dosage, See for example, Remington's Pharmaceutical Sciences,18th Ed., 1990, Mack Publishing Co., Easton, Pa. 18042 pages 1435-1712,the disclosure of which is hereby incorporated by reference in itsentirety for ail purposes and in particular for ail teachings related toformulations, routes of administration and dosages for pharmaceuticalproducts. Such formulations0 influence the physical state, stability,rate of in vivo release, and rate of in vivo clearance of theadministered agents. Depending on the route of administration, asuitable dose is calculated according to body weight, body surface areaor organ size. Appropriate dosages may be ascertained through use ofestablished assays for determining blood level dosages in conjunctionwith appropriate dose-response data. The final dosage regimen isdetermined by the attending physician, considering various factors whichmodify the action of drugs, e.g. the drug's specific activity, theseverity of the damage and the responsiveness of the patient, the age,condition, body weight, sex and diet of the patient, the severity of anyinfection, time of administration and other clinical factors. By way ofexample, a typical dose of coagulation factor complex of the presentinvention is approximately 50 IU/kg, equal to 500 μg/kg. As studies areconducted, further information will emerge regarding the appropriatedosage levels and duration of treatment for various diseases andconditions.

In some embodiments. coagulation factor complex is administered to asubject alone. In some embodiments, coagulation factor complex isadministered to a subject in combination with one or more othercoagulation factors.

In further embodiments, coagulation factor complex is administered to asubject no more than once daily. In further embodiments, coagulationfactor complex is administered to a subject: no more than once everyother day, no more than once every third day, no more than once everyfourth day, no more than once every fifth day, no more than once a week,no more than once every two weeks, no more than once a month. In stillfurther embodiments, coagulation factor complex is administered to asubject no more than twice a day.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the alterations detected in thepresent invention and practice the claimed methods. The followingworking examples therefore, specifically point out preferred embodimentsof the present invention, and are not o be construed as limiting in anyway the remainder of the disclosure.

EXAMPLES

Modifying the coagulation factors—Both FVIII and FVIIa are heavilystudied and modified molecules because of their role in the treatment ofhemophilia A. Several methods and sites on the proteins are availablefor modification. Wakabayashi, et al. (Wakabayashi, Koszelak, Mastri, &Fay, 2001) modified FVIII with acrylodan and fluorescein at Cys310 andCys692 and demonstrated that this had no effect on the activity of FVIIIin two different assays of FVIII activity. Purified FVIII can be reactedwith a maleimide compounds for one hour at room temperature or at 4° C.overnight in aqueous buffer. Maleimide reacts specifically with freecysteines and is widely used for protein labelling experiments. Labelledprotein may then be isolated via ion exchange chromatography asdescribed (Wakabayashi & Fay, 2013).

Another method to modify FVIII or FVIIa is to attach the desiredmolecule to the carbohydrate chains that modify each protein (Stennickeet at, 2013). A third method to modify either molecule is to attach adesired amino acid target to either end of the protein (Pasut &Veronese, 2012).

Albumin is known to bind a wide variety of small molecules includingbilirubin, indomethacin, ibuprofen, etc. (Peters, 1995). The binding ofmost of these ligands is not pH dependent and so can be substituted forthe fatty acid moiety at the end of the maleimide-polyethylene spacer.

Construction of the Albumin fusion proteins—The nucleotide sequence ofthe D′D3 fragment of human vWF or the soluble form of TF can be obtainedfrom Genbank. A plasmid is then constructed containing the codingsequences and an appropriate length amino acid linker. These sequencescan be flanked by appropriate DNA from the human albumin gene such thatthe construct can be inserted, in phase, at nucleotide 111 from the capsite into the human albumin gene in C3A, a human liver cell line (Kelly& Sussman, 2000). This can be accomplished using Cas/CRISPR, asdescribed (Ran et al., 2013). Additionally, a plasmid containing theappropriate sequences can be synthesized de novo.

Purification of the Albumin fusion protein—The fusion protein can berecovered from C3A cell supernate using agarose beads conjugated with amonoclonal antibody to human serum albumin. The fusion protein can thenbe eluted with human serum albumin and separated by size selectivechromatography.

Formation of the modified coagulation factor complex—The fusion proteincan be incubated at 4° C. overnight with a limiting concentration ofmodified FVIII, then purified using size selective chromatography.Similarly, the TF—albumin fusion can be incubated at 4° C. overnightwith a limiting concentration of the derivatized FVIIa, then purifiedusing size selective chromatography.

In Vivo and In Vitro Characterization—In vivo and in vitrocharacterization of the activity, pharmacokinetics and pharmacodynamicsof the therapeutic complex can be carried out as described (Mei et al.,2010; Yee et al., 2014). Unless defined otherwise, all technical andscientific terms used herein have the same meanings as commonlyunderstood by one of skill in the art to which the disclosed inventionbelongs. Publications cited herein and the materials for which they arecited are specifically incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following a claims.

Materials and Methods

Purified, B deleted recombinant Factor VIII (FVIII) was purchased fromRayBiotech (Norcross, Ga.). FVIII activity was assayed using theCoamatic FVIII assay obtained from Diapharma (West Chester Township,Ohio). FVIII ELISA was obtained from Affinity Biologicals (Ancaster, ON,CA). Other antibodies and ELISA kits were obtained from Abcam(Cambridge, Mass.). Fatty acid containing maleimide-PEG derivatives weresynthesized by Creative PEGWorks (Chapel Hill, N.C.). Othermaleimide-PEG derivatives were obtained from Nanocs (New York, N.Y.) orfrom Click Chemistry Tools (Scottsdale, Ariz.). Cell culture, molecularbiology and general reagents were obtained from Life Technologies(Carlsbad, Calif.) or SigmaAldrich (St. Louis, Mo.). Columnchromatography supplies and equipment were obtained from GE Lifesciences(Pittsburgh, Pa.).

Synthesis of CM110 and CM210—Expression plasmids for the synthetic genesCM110 and CM210 were synthesized by the Gene Art division of LifeTechnologies. The proteins were produced by polyethyleneimine (PEI)based transfection of C3A cells using a 6/1 ratio of PEI to DNA inmedium containing 5% defined calf serum. After 24 hours, the medium waschanged to serum free DMEM containing Glutamax. Medium was changed andcollected every day for four days. Supernatant medium was passed over a1 ml HisTRAP column, wash with ten column volumes of buffer containing20 mM potassium phosphate, pH 7.4, 0.5 M NaCl and 30 mM imidazole.Protein was eluted from the column with the same buffer containing 300mM imidazole. Protein containing fractions were pooled and concentrated,then changed into buffer containing 20 mM HEPES, pH 7.4, 150 mM NaCl, 4mM CaCl₂, 0.01% Tween 20, by passage over gel filtration spin columns.The protein solution was then applied to a 10×300 mm Superdex 200Increase column, run at 0.5 ml/min in the same buffer on an Akta Purechromatography apparatus. Protein containing fractions were pooled andconcentrated.

Modification of FVIII or CM210—Factor VIII was modified with a varietyof molecules of the general structure maleimide—PEGn-X, where X wasbiotin, fluorescein isothiocyanate, laurate, myristate, transcycloocteneor methyl tetrazine. Factor VIII was dissolved in buffer containing 20mM HEPES, pH 7.4, 150 mM NaCl and 4 mM CaCl₂, 0.01% Tween 20 thenincubated for one hour at room temperature with 10 μM maleimide-PEG-Xreagent. Excess reagent was removed by passage over either gelfiltration spin columns for the smaller PEG compounds or over Superdex200 Increase for the PEG reagents over 1000 daltons. CM110 or CM210 werederivatized with mal-PEG-TCO using the same conditions.

Complex formation—The FVIII/CM110 or CM210 complexes were formed byincubating modified FVIII with a ten-fold excess of CM110 or CM210 atroom temperature for one hour in buffer containing 20 mM HEPES, pH 7.4,150 mM NaCl and 4 mM CaCl₂. Complexes were purified by chromatographyover either Superdex 200 Increase or Superose 6 Increase in the samebuffer at 0.5 ml/min.

For covalent complex formation FVIII modified withmaleimide-PEG4-MeTetrazine was incubated with a ten-fold excess ofmaleimide-PEG3-TCO modified CM110 or CM210 in buffer containing 20 mMHEPES, pH 7.4, 150 mM NaCl and 4 mM CaCl2 at 4° C. overnight. Complexeswere isolated by passage over Superose 6 Increase.

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1. A coagulation factor complex comprising: a. a coagulation factor; b.a fusion protein comprising a first protein fused to albumin, or analbumin fragment; and c. a modifying molecule, wherein the modifyingmolecule is coupled to the coagulation factor in such a way as to allowbinding by the fusion protein, thereby creating a modified coagulationfactor; wherein the modified coagulation factor and the fusion proteininteract in at least two independent sites.
 2. The coagulation factorcomplex of claim 1, wherein at least one binding site of the modifiedcoagulation factor is a natural binding site.
 3. The coagulation factorcomplex of claim 1, wherein at least one binding site of the fusionprotein is provided by the modifying molecule.
 4. The coagulation factorcomplex of claim 1, wherein the coagulation factor is Factor VIII. 5.The coagulation factor complex of claim 1, wherein the modifiedcoagulation factor is modified Factor VIIa.
 6. The coagulation factorcomplex of claim 1, wherein the first protein of the fusion protein is afragment of von Willebrand's factor.
 7. The coagulation factor complexof claim 6, wherein the fragment of von Willebrand's factor is a D′D3fragment.
 8. The coagulation factor complex of claim 6, the firstprotein of the fusion protein is Tissue Factor (TF).
 9. The coagulationfactor complex of claim 1, wherein the modified coagulation factor andthe fusion protein form a non-covalant bond.
 10. The coagulation factorcomplex of claim 1, wherein the modified coagulation factor and thefusion protein interact covalently.
 11. The coagulation factor complexof claim 10, wherein the covalent bond is not a peptide bond.
 12. Thecoagulation factor complex of claim 10, wherein the covalent bond is nota peptide bond produced by translation of a nucleic acid.
 13. Thecoagulation factor complex of claim 1, wherein the modifying moleculecomprises a fatty acid.
 14. The coagulation factor complex of claim 1,wherein the modifying molecule is produced by click chemistry.
 15. Thecoagulation factor complex of claim 1, wherein the coagulation factor iscoupled to the fusion protein by click chemistry.
 16. The coagulationfactor complex of claim 1, wherein the modifying molecule is attached tothe coagulation factor through a polyethylene glycol chain. 17.(canceled)
 18. (canceled)
 19. The coagulation factor complex of claim 1,wherein the first protein of the fusion protein is joined together withalbumin via a linker.
 20. The coagulation factor complex of claim 1,wherein the coagulation factor comprises modified amino acids.
 21. Thecoagulation factor complex of claim 1, wherein the fusion proteincomprises modified amino acids.
 22. The coagulation factor complex ofclaim 1, wherein half-life of the coagulation factor complex is at least20% greater compared to a coagulation factor alone. 23-26. (canceled)